LOCUS Exported 7668 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAG413GPD-ccdB-HA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7668) TITLE S cerevisiae Advanced Gateway Destination Vectors REFERENCE 2 (bases 1 to 7668) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7668 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(10..28) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" misc_feature 70..573 /label=CEN/ARS /note="S. cerevisiae CEN6 centromere fused to an autonomously replicating sequence" promoter 610..714 /gene="bla" /label=AmpR promoter CDS 715..1575 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(933..952) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1746..2334 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2235..2254 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2488..2505 /label=L4440 /note="L4440 vector, forward primer" promoter 2658..2688 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2696..2712 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 2701..2723 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2720..2736 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2720..2736 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2755..2775 /label=T3 /note="T3 promoter, forward primer" promoter 2757..2775 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 2804..3447 /gene="S. cerevisiae TDH3" /label=GAP promoter /note="promoter for glyceraldehyde-3-phosphate dehydrogenase" primer_bind 3341..3364 /label=GPDpro-F /note="S. cerevisiae GPD promoter, forward primer" primer_bind 3453..3469 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 3453..3469 /label=pBluescriptSK /note="For pBluescript vector" protein_bind 3477..3601 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 3626..3656 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 3710..4369 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3776..3795) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 4714..5019 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 4898..4917 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(5060..5184) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 5225..5314 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" terminator 5333..5580 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(5349..5367) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" primer_bind complement(5598..5617) /label=T7 /note="T7 promoter, forward primer" promoter complement(5599..5617) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5627..5644) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5627..5643) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5636..5658) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5788..6243 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5875..5894) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6085..6106 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6503..7165) /codon_start=1 /gene="S. cerevisiae HIS3" /product="imidazoleglycerol-phosphate dehydratase, required for histidine biosynthesis" /label=HIS3 /note="yeast auxotrophic marker" /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM" promoter complement(7166..7352) /gene="S. cerevisiae HIS3" /label=HIS3 promoter primer_bind complement(7499..7518) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7618..7640 /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata 121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt 181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa 241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata 301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct 361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata 421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact 481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa 541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg 601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact 2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg 2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt 2761 aaccctcact aaagggaaca aaagctggag ctcttcgagt ttatcattat caatactcgc 2821 catttcaaag aatacgtaaa taattaatag tagtgatttt cctaacttta tttagtcaaa 2881 aaattagcct tttaattctg ctgtaacccg tacatgccca aaataggggg cgggttacac 2941 agaatatata acatcgtagg tgtctgggtg aacagtttat tcctggcatc cactaaatat 3001 aatggagccc gctttttaag ctggcatcca gaaaaaaaaa gaatcccagc accaaaatat 3061 tgttttcttc accaaccatc agttcatagg tccattctct tagcgcaact acagagaaca 3121 ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga gtgatgcaac ctgcctggag 3181 taaatgatga cacaaggcaa ttgacccacg catgtatcta tctcattttc ttacaccttc 3241 tattaccttc tgctctctct gatttggaaa aagctgaaaa aaaaggttga aaccagttcc 3301 ctgaaattat tcccctactt gactaataag tatataaaga cggtaggtat tgattgtaat 3361 tctgtaaatc tatttcttaa acttcttaaa ttctactttt atagttagtc ttttttttag 3421 ttttaaaaca ccagaactta gtttcgacgg attctagaac tagtggatcc cccatcacaa 3481 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt 3541 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat 3601 ggcggccgca ttaggcaccc caggctttac actttatgct tccggctcgt ataatgtgtg 3661 gattttgagt taggatccgt cgagattttc aggagctaag gaagctaaaa tggagaaaaa 3721 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc 3781 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt 3841 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc 3901 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat 3961 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc 4021 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt 4081 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt 4141 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga 4201 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct 4261 gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg tcggcagaat 4321 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa cgccgcgtgg 4381 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat 4441 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag 4501 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca 4561 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac 4621 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg 4681 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa 4741 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg 4801 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa 4861 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc 4921 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac 4981 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac 5041 agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt attatgtagt 5101 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg 5161 ttcagctttc ttgtacaaag tggtgatggg ctgcaggaat tcgatatcaa gcttaggtgg 5221 aatgtaccca tacgatgttc ctgactatgc gggctatccc tatgacgtcc cggactatgc 5281 aggatcctat ccatatgacg ttccagatta cgctgctcag tgctaactcg agtcatgtaa 5341 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa 5401 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta 5461 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt 5521 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc 5581 ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 5641 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 5701 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 5761 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg 5821 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5881 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5941 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 6001 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 6061 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 6121 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 6181 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 6241 tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatagatcc 6301 gtcgagttca agagaaaaaa aaagaaaaag caaaaagaaa aaaggaaagc gcgcctcgtt 6361 cagaatgaca cgtatagaat gatgcattac cttgtcatct tcagtatcat actgttcgta 6421 tacatactta ctgacattca taggtataca tatatacaca tgtatatata tcgtatgctg 6481 cagctttaaa taatcggtgt cactacataa gaacaccttt ggtggaggga acatcgttgg 6541 taccattggg cgaggtggct tctcttatgg caaccgcaag agccttgaac gcactctcac 6601 tacggtgatg atcattcttg cctcgcagac aatcaacgtg gagggtaatt ctgctagcct 6661 ctgcaaagct ttcaagaaaa tgcgggatca tctcgcaaga gagatctcct actttctccc 6721 tttgcaaacc aagttcgaca actgcgtacg gcctgttcga aagatctacc accgctctgg 6781 aaagtgcctc atccaaaggc gcaaatcctg atccaaacct ttttactcca cgcacggccc 6841 ctagggcctc tttaaaagct tgaccgagag caatcccgca gtcttcagtg gtgtgatggt 6901 cgtctatgtg taagtcacca atgcactcaa cgattagcga ccagccggaa tgcttggcca 6961 gagcatgtat catatggtcc agaaacccta tacctgtgtg gacgttaatc acttgcgatt 7021 gtgtggcctg ttctgctact gcttctgcct ctttttctgg gaagatcgag tgctctatcg 7081 ctaggggacc accctttaaa gagatcgcaa tctgaatctt ggtttcattt gtaatacgct 7141 ttactagggc tttctgctct gtcatctttg ccttcgttta tcttgcctgc tcatttttta 7201 gtatattctt cgaagaaatc acattacttt atataatgta taattcatta tgtgataatg 7261 ccaatcgcta agaaaaaaaa agagtcatcc gctaggggaa aaaaaaaaat gaaaatcatt 7321 accgaggcat aaaaaaatat agagtgtact agaggaggcc aagagtaata gaaaaagaaa 7381 attgcgggaa aggactgtgt tatgacttcc ctgactaatg ccgtgttcaa acgatacctg 7441 gcagtgactc ctagcgctca ccaagctctt aaaacgggaa tttatggtgc actctcagta 7501 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cgcgctgacg 7561 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 7621 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcga //